Cloning and in vitro antiapoptotic effects of pig FLIPs.
BACKGROUND
Cellular FLICE-like protein (cFLIP) inhibits death receptor-mediated apoptosis signal transduction, such as that induced by Fas and TNFR. The present study examined the role of antiapoptotic molecules to protect pig cells from human natural killer (NK) cells in vitro, as a model of delayed-type xenograft rejection.


METHODS
Pig FLIPs were cloned using the TBLASTIN program to search for cDNA fragments of pig FLIPs. The sequence was identified using the dideoxy chain termination method and an ABI PRISM3100 genetic analyzer. The cDNA of pig FLIPs was inserted into the cloning site of the chicken beta-actin promoter (pCXN2). The cDNA was then transfected into pig endothelial cells (PEC), to establish several stable PEC clones containing the cDNA. Expression of the pig FLIP gene was evaluated by reverse-transcriptase polymerase chain reaction, and NK cell-mediated cytolysis assessed, using YT cells (an NK-like cell line).


RESULTS
The full-length pig FLIP encoding sequence, total 5'-region to 3'-region, was defined for the first time. PEC transfectants with the FLIP showed moderate expression of FLIPs. Transfection of PEC with plasmids encoding FLIPs inhibited NK cell-mediated PEC lysis. While approximately half of parental PEC were injured by the human NK-like YT cells, the injury rate was relatively lower in the transfectants.


CONCLUSIONS
Overexpression of the antiapoptotic molecules, pig FLIPs, has the potential for use in protecting graft cells from human NK cells.
Xu
H
H
Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan.
Firdawes
S
S
Yamamoto
A
A
Matsunami
K
K
Ishimaru
A
A
Kondo
A
A
Fukuzawa
M
M
Miyagawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005089:Exocytosis; D007694:Killer Cells, Natural / Q000276:immunology / Q000502:physiology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D018076:DNA, Complementary; D012333:RNA, Messenger